FISH Analysis of Chromosomes and DNA Recombination

7/29/2019 FISH Analysis of Chromosomes and DNA Recombination

1/32

FISH analysis of chromosomes:

Fluorescent IN SITU hybridization


2/32

metaphase spread chromosomes stained with

DAPI, a fluorescing stain that specifically binds

double stranded DNA


3/32

green = probe for end of chromosome 4

Expose DAPI-stained metaphase

chromosomes to fluorescent probes

red = control probe for centromere of

the X chromosome & another probe forend of chromosome X


4/32

DiGeorge syndrome/CATCH22microdeletion on chromosome 22

birth defect that affects the immune system

absence of or underdevelopment of the thymus and

parathyroid glands

facial features include low-set ears, wide-set eyes, small

jaw, and bowing up of upper lip


5/32

FISH tests: DiGeorge syndrome

Expose DAPI-stained

chromosomes to mixture

of fluorescent probes

green = control probe for

chromosome 22

red = probe for DiGeorge

region on long arm of

chromosome 22


6/32

FISH tests: Painting chromosomes

Expose chromosomes to fluorescent probes that highlight

entire chromosomes


7/32

FISH tests: Painting chromosomes

Expose chromosomes to fluorescent probes that highlight

chromosomes 13, 18, 21, X, and Y

nuclei fromthe same

fetus

green = chromosome 13

red = chromosome 21

aqua = chromosome 18

green = X chromosome

red= Y chromosome


8/32


9/32

DNA Recombination

General recombination

Site specific recombination


10/32

General DNA Recombination


11/32

Heteroduplex joint


12/32

General Recombination

Two homologous DNA molecules cross over

The site of exchange can occur anywhere

A strand of one DNA molecule has become

base-paired to a strand of the second DNA

to create heteroduplex joint

No nucleotide sequences are altered


13/32

The procedure of general recombination

DNA synapsis: base pairs form between

complementary strands from the two DNA molecules


14/32

DNA Hybridization

The initial step for DNArecombination


15/32

RecA protein-mediated DNA synapsis

Rec A has multiple DNA binding sites, hence can hold a single strand

and a double helix together

Rec A is also a DNA-dependent ATPase


16/32

DNA Branch Migration


17/32

Holiday Junction for DNA recombination

Exchange of the first single strand between two different DNA double helices is slow and

difficult, then intermediate state Holiday Junction, then complete exchange


18/32


19/32

Holiday Junction for DNA

recombinationand its resolution


20/32

Summary for General

RecombinationGeneral recombination allows large fraction of

genetic information to move from one

chromosome to another.

General recombination requires the breakage ofdouble helices, beginning with a single strand

breakage.

General recombination is facilitated by Rec A inbacteria and its homologs in eucaryotes.

Holiday junction is the intermediate state of

general recombination


21/32

Site-specific recombinationMoves specialized nucleotide sequence (mobile

genetic elements) between non-homologous sites

within a genome.

Transpositional site-specific recombination

Conservative site-specific recombinatinon


22/32

Transpositional site-specific

recombinationModest target site selectivity and insert mobile

genetic elements into many sites

Transposase enzyme cuts out mobile genetic

elements and insert them into specific sites.


23/32

Three of the many types of mobile genetic elements found in bacteria

Transposase gene: encoding enzymes for DNA breakage and joining

Red segments: DNA sequences as recognition sites for enzymes

Yellow segments: antibiotic genes


24/32


25/32

Cut and Paste Transposition

DNA-only


26/32

The structure of the central intermediate formed by transposase (integrase)


27/32

Replicative Transposition


28/32

Retrovirus-based Transposition

Retroviral-like retrotransposition


29/32


30/32

Reverse Transcriptase

RNA


31/32

Non-retroviral retrotransposition


32/32

Conservative Site Specific Recombination

Integration vs. inversion

Notice the arrows of directions

Documents

FISH Analysis of Chromosomes and DNA Recombination